Optimization of the PBMNC reprogramming protocol.

<p>(<b>A</b>) Depletion of CD3⁺ and 19⁺ lymphoid cells increases reprogramming efficiency. Whole PBMNCs or CD3⁻/CD19⁻ cells (T/B cell-depleted PBMNCs) were cultured for 4 days before nucleofection with episomal vectors expressing OS (OCT4 and SOX2), MK (MYC and KLF4) and B (BCL-XL). 1×10⁶ cells were used for nucleofection. The numbers of iPSC colonies were counted at 3–4 weeks after nucleofection and numbers of iPSC colonies per 1 ml of PB were calculated by normalization to the amount of starting peripheral blood. Data shown are presented as mean ± SEM (n = 4). * indicates <i>P</i><0.05. (<b>B</b>) Culturing PB CD3/19⁻ cells for 4 days allows maximum reprogramming. CD3/19⁻ cells (T/B cells-depleted) PBMNCs were cultured for 0 to 8 days before nucleofection with episomal vectors expressing OS, MK and B. 1×10⁶ cells were used for nucleofection. The numbers of iPSC colonies were counted at 3–4 weeks after nucleofection. Graphed data are presented as mean ± SEM (n = 6). * indicates <i>P</i><0.05.</p

BCL-XL significantly enhances OS-mediated reprogramming of cord blood and peripheral blood cells with lentiviral vectors.

<p>(<b>A</b>) Differential effects of BCL2 family members on enhancing OS-mediated reprogramming of CB cells. CB CD34⁺ cells were cultured for 2 days before lentiviral transduction. CB iPSC colonies were enumerated at 2 weeks after transduction of reprogramming factors. Data shown are presented as mean ± SEM (n = 4). OS: OCT4 and SOX2. * indicates <i>P</i><0.05. (<b>B</b>) Differential effects of BCL2 family members on enhancing OS-mediated reprogramming of PBMNCs. PBMNCs were cultured for 4–6 days before lentiviral transduction. PB iPSC colonies were enumerated at 3 weeks after transduction of reprogramming factors. Data shown are presented as mean ± SEM (n = 4). OS: OCT4 and SOX2. PBMNCs, peripheral blood mononuclear cells. * indicates <i>P</i><0.05. BCL2 and BCL-XL significantly increased reprogramming of both CB CD34⁺ cells and PBMNCs.</p

In vitro multilineage differentiation of integration-free PB iPSCs.

<p>(<b>A</b>) Differentiation of PB iPSCs into MSCs. iPSC-MSCs show a typical MSC morphology and are capable of differentiation into adipocytes, osteoblasts and chondrocytes. Oil Red O stains the oil droplets of adipocytes. Alizarin Red stains the bone nodules formed by osteoblasts. Alcian Blue stains acid mucopolysaccharides synthesized and secreted by chondrocytes. (<b>B</b>) PB iPSC-derived hepatocytes show a typical morphology of hepatocytes at 25 days after hepatocytic differentiation. These cells also express markers AFP, albumin (ALB), and alpha 1-antitrypsin (α1-AT). The differentiated cells were stained with monoclonal antibody against AFP, goat anti-albumin, and goat anti-alpha 1-antitrypsin at 18 days after differentiation culture. (<b>C</b>) PB iPSC can be induced to differentiation into cardiomyocytes that express Troponin I marker. Cell nuclei were counterstained with DAPI.</p

Generation of integration-free iPSCs from adult PBMNCs with episomal vectors.

<p>(<b>A</b>) ALP staining of iPSCs at 4 weeks after nucleofection of PBMNCs with reprogramming factor-expressing episomal vectors. OS, OCT4 and SOX2; MK, MYC and KLF4; B, BCL-XL. PBMNCs were cultured for 4–8 days before nucleofection. 1×10⁶ PBMNCs were nucleofected and then seeded into each well. (<b>B</b>) Inclusion of BCL-XL increases PB reprogramming efficiency by up to 10-fold. PBMNCs were cultured for 4–8 days before nucleofection. ALP-positive iPSC colonies were enumerated at 3–4 weeks after nucleofection. Data are presented as mean ± SEM (n = 6). In all 3 conditions, BCL-XL significantly increased reprogramming efficiency. * indicates <i>P</i><0.05. (<b>C</b>) iPSC are generated from PBMNCs expressing the myeloid lineage marker, CD33, but not lymphoid cells (CD3⁺ and CD19⁺ cells) in PBMNCs. ALP staining of iPSCs at 4 weeks after nucleofection of fractionated PBMNCs with episomal vectors OS+MK+B. CD33, myeloid marker; CD3, T cell marker; CD19, B cell marker. 1×10⁶ indicated cells were nucleofected and then seeded into each well. ALP staining was conducted at 4 weeks after nucleofection.</p